AIM： To examine the role of Fibrinogen-like protein 2 （fgl2）/fibroleukin in tumor development. Fgl2 has been reported to play a vital role in the pathogenesis in MHV-3 （mouse hepatitis virus） induced fulminant and severe hepatitis, spontaneous abortion, allo- and xenograft rejection by mediating ＂immune coagulation＂.METHODS： Tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma （HCC） model on nude mice （established from high metastasis HCC cell line MHCC97LM6） were obtained. RESULTS： HfgI2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human HCC nude mice. Hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, NK cells, and CD8^＋ T lymphocytes and vascular endothelial cells. HfgI2 mRNA was localized in cells that expressed hfgI2 protein. Fibrin （nogen） colocalization with hfgl2 expression was determined by dual immunohistochemical staining. In vitro, IL-2 and IFN-γ, increased hfgl2 mRNA by 10-100 folds and protein expression in both THP-1 and HUVEC cell lines. One-stage clotting assays demonstrated that THP-1 and HUVEC cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. CONCLUSION： The hfgI2 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction.
Kai Su Fang Chen Wei-Ming Yan Qi-Li Zeng Li Xu Dong Xi Bin Pi Xiao-Ping Luo Qin Ning
Previous study on TNFRl-mediated hepatocyte apoptosis has been implicated in the development of fulminant viral hepatitis. To interfere with the potentially effective target, plasmid named p-mTNFRlshRNA complimentary to the sequence responsible for mTNFR1 was also constructed and further confirmed by sequence analysis. To investigate the effect of mTNFRlshRNA plasmid on mTNFR1 expression in vivo and the disease progress in MHV-3 induced fulminant hepatitis mice model. By hydrodynamic injection of mTNFRlshRNA plasmid, the survival rate of mice, hepatic pathological change were examined and compared between mice with/without mTNFRlshRNA plasmid intervention. The expression of mTNFR1 was detected by Real-time PCR, immunohistochemistry staining. The mTNFRlshRNA plasmid significantly reduced mTNFR1 expression in vivo, markedly ameliorates inflammatory infiltration, prolonged the survival time period and elevated the survival rate from 0 up to 13.3% in Balb/cJ mice with MHV-3 induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in inhibiting TNFR1 expression, which has been reported to be involved in the development of a variety of diseases including fulminant viral hepatitis and severe chronic hepatitis B.
Sui GAO Ming WANG Jian-wen GUO Dong Xi Xiao-ping LUO Qin NING
To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type 3(MHV-3) induced chronic viral hepatitis in C3H/Hej mice,ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units(PFU)of MHV-3 intraperitoneally.The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin(HE) staining method from 2 days post MHV-3 infection.The ratios of T cell subsets including CD3+CD4+CD8-,CD3+CD4-CD8+,CD3+CD4-CD8-,CD3+CD4+CD25+,CD3+CD4+CD25-and CD3+ CD4-CD25+ T lymphocyte of total T lymphocytes in blood,spleen and liver were examined at 0,2,4,6,8,10,12,15,20,25,30,40 days post MHV-3 infection by flow cytosorting.We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection.The double negative T cell(DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice,and CD3+CD4+CD8-,CD3+CD4-CD8+,CD3+CD4+CD25-and CD3+CD4-CD25+ T cell ratios decreased accordingly.In conclusion,the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence.Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection.Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.
Jiang-guo ZHANG Xiao-min QIN Xiao-jing WANG Wei-ming YAN Chuan-long ZHU Xiao-ping LUO Qin NING
This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2(mfgl2),which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis.A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed,while another short hairpin RNA(shRNA) which was a mutated form of the mfgl2shRNA sequences was used as a control.A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression.By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells,the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy,FACS,RT-PCR and immunohistochemistry staining.The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%.The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.
Dong XI Zhi-Mo WANG Sui GAO Chuan-Long ZHU Jian-Wen GUO Xiao-Ping LUO Qin Ning
The role of chemokines in murine hepatitis virus strain 3 (MHV-3) induced fulminant hepatic failure (FHF) is not well defined. In this study, we investigated the role of the CXC chemokine receptor 3 (CXCR3)- associated chemokine [monokine induced by IFN-gamma (Mig/CXCL9) and interferon-gamma-inducible protein 10 (IP-10/CXCL10)] in the recruitment of intrahepatic lymphocytes and subsequent fulminant hepatic failure induced by MHV-3. Balb/cJ mice (6-8 weeks, female) were intraperitioneally injected with 100 PFU MHV-3.The proportions and numbers of T cells and NK cells as well as the expression of CXCR3 on T cells and NK cells in the liver, spleen and blood were analyzed by flow cytometry. The hepatic mRNA level of the CXCR3-associated chemokines (CXCL9 and CXCL10) was detected by realtime PCR. A transwell migration assay was used to assess the chemotactic effect of MHV-3-infected hepatocytes on the splenic lymphocytes. Following MHV-3 infection, the number of hepatic NK cells and T cells and the frequencies of hepatic NK cells and T cells expressing CXCR3 increased markedly; however, in the spleen and peripheral blood, they both decreased significantly. Moreover, the hepatic mRNAs levels of CXCL9 and CXCL10 were significantly elevated post infection. The transwell migration assay demonstrated that MHV-3-infected hepatocytes have the capacity to attract and recruit the splenic NK cells and T cells, and CXCL10 plays a key role in lymphocyte mobilization from the spleen. These results suggest that the CXCR3- associated chemokines (CXCL9 and CXCL10) may play animportant role in the recruitment of intrahepatic lymphocytes and subsequent necroinflammation and hepatic failure in MHV-3 infection.
Yong ZOUGe SONGLin DINGTao CHENHong-wu WANGWei-ming YANXiao-jing WANGXiao-ping LUOQin NING
